TR201701544A2 - - Google Patents

Abstract

The present invention relates to a plant exosome-containing product which can be used in the treatment of cancer and wound healing. With the invention, a cost-effective product is obtained which does not cause toxic effects on the human body, does not cause any side effects in the treatment of chemotherapy, does not cause damage to healthy cells during the cancer treatment process, does not require any procedure such as radiotherapy or surgery and does not cause any infection. Wheatgrass, garlic and ginger can be used singly or in combination as vegetable source in the invention.

Description

DESCRIPTION A PRODUCT CONTAINING A VEGETABLE EXOZOME Technical Area This invention is a herbal exosome that can be used in cancer treatment and wound healing. Prior Art In multicellular organisms, proteins secreted from the cell communication, which is carried out as a result of binding to the receptors on its surface, It is the basic principle of intercellular communication. Unless you have a lot of information about At the beginning of the communication molecules, which are in great demand today, cksosomes coming (1-3). material unwanted by the cell when first discovered Although it is named as waste, as a result of researches, exosomes It has been proven to have a major role in the immune system (4). Exosomes 20-130 Plasma by all cells between nm in size (in nanoscale) They are vesicles that are produced inside the cell and secreted out of the cell (5). Soon As it has been discovered, these vesicles are autocrine and distal to local or distal host cells. It plays a role in the endocrine system to provide paracrine signals. These cksosomes are the same also by transporting protein, fat, nucleic acid materials (all types of RNA) and they are responsible for transferring them to the recipient cells. In this way, the cell Messages to be transported will be transferred to other neighboring cells (3). Tetraspanin family is the most common group of proteins on the exosome surface. in this family There are CD9, BCD63, CD81, CD82, CD83 proteins. tetraspanin family proteins have the ability to interact with MHC and integrin proteins (6).

No matter how generalized, the exosome protein pool is more cell-specific. It depends on the cell from which the exosome is released. Therefore, various (heat- 25418.61 shock) proteins are among the members of this pool (hsp70). At the same time the cell proteins in the skeleton, ß actin, tubilin, myosin, cofilin, basic tissue- Compatibility component class I-II molecules and glyceraldehyde 3 phosphate dehydrogenation It is one of the main common proteins of the exosome protein pool. The exosome will be transmitted stimulating the surface receptors of the other cell so that it can be taken into the cell required. For this reason, Wnt-ß-catenin signaling proteins, notchligang, delta-like 4 proteins and interleukins must be present on the exosome surface. Opposite Otherwise, it will not be possible for the exosome to stimulate the host cell to which it will transmit a message (7,8,9).

In his study, exosomes obtained from mammalian cells were found to be It has been proven to have the potential to be used in the treatment (Raghu et al. 2016). In addition, exosomes are anticarcinogenic on breast cancer cell. It has been shown to have an effect(0hn0 et al. 2013). Another similarly executed In this study, the effect of exosomes obtained from brain epithelium on brain cancer. (Reriro et al. 2015).

Wheat (Triticum Aestivum) is an important plant food in human diet. plays a role. In line with the epidemiological studies, chronic It has been proven to play a protective role in thalassemia and cancer patients. your wheat many forms (wheatgrass or seeds) to be used in cancer treatment developed. Likewise, ginger and garlic have antioxidant properties. made Exosomes of animal origin are used for different applications in their studies. is used. In addition, plant-derived exosomes are antioxidants. Although it has been identified, there is a great deficit in its study. has not been observed. Plant exosomes, as far as is known, have been associated with mammalian exosomes. has similar features.

Targeting dividing cells such as surgery, radiotherapy and chemotherapy treatment methods can result in recurrence of cancer many times or the occurrence of many side effects. results in the output (Kubota, 2012). 25418.61 from exosome cells isolated from healthy cells for use is mentioned.

With the invention, with the product containing herbal exosomes, cancerous cells without damaging the healthy cells It has been developed as an alternative treatment method to cause the death of cells.

In addition, the fact that exosomes are cell-free products means that cellular therapy is It has revealed the situation of preventing the problems that may arise.

Exosomes are ready-to-use therapeutics. Toxic cryocondom agent and ultra- It can be stored at low temperatures without the need for a freezer and can be reached with confidence. Based on this information, wheat used in alternative type The use of grass, garlic and ginger in cancer treatment and wound healing is targeted.

Brief Description of the Invention The aim of the invention is to produce a herbal exosome-derived product that can be used in cancer treatment. is to obtain.

Another object of the invention is to obtain a product that does not produce a toxic effect on the human body.

Another purpose of the invention; an effective product in wound healing, repair of damaged tissue is to provide.

Another purpose of the invention; side effects of chemotherapy is to obtain a product that has not been experienced.

Another purpose of the invention; cause damage to healthy cells during cancer treatment is to obtain a product that does not open. 25418.61 Another purpose of the invention; any procedure, such as radiotherapy or surgery It is to obtain a product that does not pose an infection risk because it does not require

Another purpose of the invention; to obtain a low-cost product.

Another purpose of the invention; chemical in the body caused by synthetic drugs is to obtain a natural product without contamination.

Another purpose of the invention; The plants to be used in the treatment grow easily and It is a practical and easily accessible product as exosomes are obtained on a large scale in a short time. is to provide.

Detailed Description of the Invention With a "Product Containing Herbal Exosome" developed to achieve the purpose of the invention The relevant figures are shown in the appendix; Figure 1-A: Control group flow cytometry values of A498 kidney cancer cells is the view.

Figure 1-B: Flow of A498 kidney cancer cells with control group Annexin V stain values are cytometry view. 25418.61 Figure 1-C: Flow of A498 kidney cancer cells with control PI stain cytometry values view.

Figure 1-D: 15 mg/ml wheat exosome was applied to A498 kidney cancer cells. Flow cytometry is the view of death values.

Figure 1-E: 15 mg/ml ginger exosome of A498 kidney cancer cells Applied flow cytometry measurement values view.

Figure 1-F: 15 mg/ml garlic exosome of A498 kidney cancer cells Applied flow cytometry measurement values view.

Figure 2-A: Control group flow cytometry of HEK 293 kidney epithelial cells values view.

Figure 2-B: Flow of HEK 293 kidney epithelial cells with control group Pl stain cytometry values are sent.

Figure 2-C2HEK 293 Control group of kidney epithelial cells with Annexin V stain Flow cytometry values view.

Figure 2-D: HEK 293 15 mg/ml wheat exosome of kidney epithelial cells Applied flow cytometry measurement values view.

Figure 2-E: HEK 293 15 mg/ml ginger exosome of kidney epithelial cells Applied flow cytometry measurement values view.

Figure 2-F: HEK 293 15 mg/ml garlic exosome of kidney epithelial cells Applied flow cytometry measurement values view.

Figure 3-A: Control group flow cytometry values of 22RV1 prostate cancer cells is the view. 25418.61 Figure 3-B: Control group of 22RV1 prostate cancer cells with Annexin V stain Flow cytometry values view.

Figure 3- Flow of Cs22RV1 prostate cancer cells with control PI stain cytometry values view.

Figure 3-D: 15 mg/ml wheat exosome of 22RV1 prostate cancer cells Applied flow cytometry measurement values view.

Figure 3-E: 15 mg/ml ginger exosome of 22RV1 prostate cancer cells Applied flow cytometry measurement values view.

Figure 3-F: 15 mg/ml garlic exosome of 22RV1 prostate cancer cells Applied flow cytometry measurement values view.

Figure 4-A: Control group flow cytometry values of PNTlA prostate epithelial cells is the view.

Figure 4-B: Flow of PNT1A prostate epithelial cells with control PI stain cytometry values view.

Figure 4-CiPNT1A control group of prostate epithelial cells flow with Annexin V stain cytometry values view.

Figure 4-D: 15 mg/ml wheat exosome of PNTlA prostate epithelial cells Applied flow cytometry measurement values view.

Figure 4-E: 15 mg/ml ginger exosome of PNTlA prostate epithelial cells Applied flow cytometry measurement values view.

Figure 4-F: 15 mg/ml garlic exosome of PNTlA prostate epithelial cells Applied flow cytometry measurement values view. 25418.61 Figure 5-A: Control group flow cytometry values of MCF 7 breast cancer cells is the view.

Figure 5-B: Flow of MCF 7 breast cancer cells with control group Annexin V stain cytometry values view.

Figure 5-C2MCF 7 breast cancer cells flowing with control group P1 stain cytometry values view.

Figure 5-D: 15 mg/ml wheat exosome of MCF 7 breast cancer cells was applied Flow cytometry is the view of death values.

Figure 5-E: 15 mg/ml ginger exosome of MCF 7 breast cancer cells Applied flow cytometry measurement values view.

Figure 5-F: 15 mg/ml garlic exosome of MCF 7 breast cancer cells Applied flow cytometry measurement values view.

Figure 6-A: Control group flow cytometry of MCF 10A breast epithelial cells values view.

Figure 6-B: Flow of MCF 10A mammary epithelial cells with control PI stain cytometry values view.

Figure 6-C:MCF 10A control group of breast epithelial cells with Annexin V stain Flow cytometry values view.

Figure 6-D: 15 mg/ml wheat exosome of MCF 10A mammary epithelial cells Applied flow cytometry measurement values view.

Figure 6-E: 15 mg/ml ginger exosome of MCF 10A mammary epithelial cells Applied flow cytometry measurement values view. 25418.61 Figure 6-F: 15 mg/ml garlic exosome of MCF 10A mammary epithelial cells Applied flow cytometry measurement values view.

Figure 7- The presence of plant exosome obtained from the wheat plant was determined. Electron Microscope view.

Figure 8- The presence of plant exosome obtained from wheat plant on HSP 70 surface is the flow cytometry view in which the marker is detected.

In line with these results, plant exosome only transforms cancer cells into apoptosis. It causes cell death by causing cell death, but in healthy cells, any cell death occurs. It has not been shown to cause death.

Experimental Study Preparation of Plant Originated Exosome Wheatgrass as a plant source in the product containing herbal exosomes, which is the subject of the invention, Ginger and garlic can be used alone or in combinations.

Wheatgrass, preferably selected in experimental studies, Turkey, Adana Ceyhan 69 obtained from seeds. In the preliminary sample preparation studies, It was determined that the seeds were sufficient to grow in 1.5 weeks.

The collected grass was first ground in %lalic PBS (Phosphate Buffered Salt). Afterwards, the filtering process was carried out. The filtrate obtained from wheatgrass 10 culture (cellculture) was isolated using exosome isolation kit. isolated exosomes were dissolved in 0.9% isotonic serum. 25418.61 The product of the invention, which is isolated and in the form of serum, exosomes, scanned observed using electron microscopy (Figure 7). HSP 70 marker after Observation of the presence of exosomes in flow cytometry by incubating with has been made.

Development of Treatment Determination of Cell Toxicity The toxic effect of the prepared product, which is the subject of the invention, is determined by the MTS method in the literature. (Yalvac et al, 2009).

Chemical molecules were prepared in the medium at the specified concentrations and 96 HEK seeded by counting (5000 cells/well) in well-cultured culture dishes (human kidney epithelial cell), MCF 10A (human mammary epithelial cell), PNTlA(human prostate epithelial cell) was applied on cell lines. against chemicals response to toxicity in cells, in the form of measuring cell viability for 4 days has been detected. Cell viability analysis was performed according to the MTS method. MTS, It is a colorimetric method used to measure mitochondrial enzyme activity.

By applying different doses to the cells previously seeded in the wells, The effect of the applied substance on the vitality within the specified time interval has been examined. MTS agent given by mixing with the media, the number of viable cells increases, it acquires a dark color. The resulting color change using the ELISA reader It was evaluated as absorbance measurement. The invention was developed under The product is found in healthy cells at concentrations of 5ng/ml, 10 ng/ml, 15 ng/ml. It is prepared to get results at 24, 48, 72 and 96 hours. 25418.61 Cancer Cell Death Analysis In order to examine the effect of the product of the invention on cancer, the suicide mechanism Annexin V protein, one of the most prominent proteins of the apoptosis pathway, existence is checked. Presence of necrosis with Pl (Propidium lodide) stain has been taken care of. 22RV1 (prostate cancer cell), MCF 7 (breast cancer cell), A498 (kidney cancer cell) cells in 6-well culture dishes at 300,000 cells/well will be planted as it will be. RPMI for cell line 22RV1, cell line MCF 7 and A498 For DMEM high glucose media, 10% PES (FetalBovine Serum) and 1% PSA (Penicillin Streptomycin Amphotericin) was added to ensure the growth of cells.

The death rates of cells treated at the indicated concentrations were determined after 24 hours. has been examined. The cell that binds to the Annexin V antibody undergoes apoptosis or that the cell bound to the Pl antibody has gone into necrosis or It indicates that you are about to leave. Based on the presence of these antibodies It has been determined that the treatment kills the cell.

Same experimental protocol as healthy cell lines MCF10-A, PNT1-A and HEK cell lines. applied to the lines.

Examination of wound healing In addition to the use of the product of the invention in the treatment of cancer, Its use in healing has also been studied. In this experimental study, The healthy cell line HEK cells were seeded in 6-well culture dishes.

Wound model is made by making a scratch that will pass right through the middle of the wells. has been created. Exosome-containing product to the cells in which the wound model is created. has been applied. Cells were grown in DMEM media containing 2% FBS at 15 ng/ml. was incubated in the concentrated product for 48 hours. everyday in the microscope The wound healing process was observed step by step by taking a photograph. 25418.61 Experimental Results With the developed isolation method, exosomes are obtained preferably using wheatgrass. has been made. This product has been subjected to characterization experiments for cancer and wound The effectiveness of its healing properties has been proven by the results. electron microscope The data obtained in the image of the visible vesicles are exosomes found in the literature. It was found to be similar to electron microscope images. (Figure 7) Flow The results obtained in the cytometry expressed the surface marker in 84.16% of the exosomes. shows it does. This means that the molecule we obtained is an exosome. (Figure 8).

When healthy cells are exposed to plant exosome serum, It has been proven by the result of MTS that it causes proliferation. According to negative control The increased absorbance amount indicates that the vitality has increased. in this direction The analyzes determined the rate of division in healthy cells of the wheat exosome. has proven to increase (Figure 3). 22RV1 cells treated with serum at a concentration of Sug/ml, 10 tig/ml, 15 tig/m1 Annexin V method to observe cell death 24 hours after was performed using flow cytometry. In line with these results, necrosis and When the apoptosis rates are calculated, the concentration specified is 26.02%, 40.08%, respectively. and 34.36% cell death was observed (Figure-3).

Sag/ml of PNTIA cells with serum at a concentration of 10 µg [ml, 15 pg /ml Annexin V to monitor cell death 24 hours after treatment method was applied using flow cytometry. In line with these results, necrosis and when the apoptosis rates are calculated, they are 1.81%, 326% and 3.22%, respectively. cell death was observed (Figure-4). 25418.61 Treatment of MCF-7 cells with serum at a concentration of Sug/ml, 10 tig/ml, 15 tig/ml Annexin V method to observe cell death 24 hours after It was performed using flow cytometry. In line with these results, necrosis and cell death was observed (Figure-5).

Serum of MCF-10A cells at a concentration of Sug/ml, 10 tig/ml, 15 tig/m1 Annexin V to monitor cell death 24 hours after treatment method was applied using flow cytometry. In line with these results, necrosis and when the apoptosis rates are examined, they are 1.57%, 125% and 274%, respectively. cell death was observed (Figure-6).

Treatment of A498 cells with serum at a concentration of Sug/ml, 10 µg /ml, 15 µg /ml Annexin V method to observe cell death 24 hours after was performed using flow cytometry. In line with these results, necrosis and cell death was observed (Figure-1).

Serum of HEK 293 cells at a concentration of Sag/ml, 10 tig /ml, 15 tig /ml Annexin V to monitor cell death 24 hours after treatment method was applied using flow cytometry. In line with these results, necrosis and apoptosis rates were 1.79%, 163% and 3.16%, respectively. cell death was observed (Figure-2).

As a result of the experimental results; suicide of cells in the body of the product of the invention It has been proven to cause death in cancer cells by triggering the mechanism of action. This cancer, which is frequently encountered in chemotherapy treatments of treatment with the product. while killing the cell, it also has the disadvantage of damaging healthy cells. appears to have been eliminated. 25418.61 As a result of the wound model created from the MTS result, the invention of the wound pattern in cells treated with the subject product It has been proven to show much faster recovery compared to control cells. This is also achieved Wound healing and tissue repair of the wheat exosome-derived product It has been shown that models affect and accelerate the process positively.

Application of the Invention The invention in question is primarily used in prostate cancer, breast cancer, kidney cancer. It is also applied to the pancreas, liver, bone, skin, brain, lung, lung. membrane, uterus, ovary, stomach, intestine, bladder, blood, lymph, thyroid cancer types. is active.

The product of the invention can be solid or hydrogel, primarily in the form of serum in liquid form. can also be produced. These are also in the form of serum, syrup, tablet, medicine, gel, cream. can happen. At the same time, it can be used as a nano carrier molecule, into the cell. another active substance (such as medicine, chemical) can be transported. This transport method can be used in different forms and formulations as stated above. 25418.61 References . Johnstone, RM, Adam, M, Hammond, JR, Orr L and Turbide, C (1987). Vesicle formation during reticulocyte maturation. Association of plasma membrane . Valadi, H, Ekström, K, Bossios, A, Sjöstrand, M, Lee JJ and Lçtvall JO (2007). Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic Exchange between cells. Nat Cell Bi0I92654-659 . Théry, C, Ostrowski, M and Segura, E (2009). Membrane vesicles as conveyors of immune responses. NatRevlmmunol 9: 581-593 . Gogolak P, Rethi B, Hajas G, Rajnavolgyi E.(2003) Targeting dendritic cells . An, Q, Hücvkelhoven, R, K0gel, KHandvan Bel, Aj(2006). multi vesicular bodies participate in a cell wall-associated defense response in barley leaves attacked by the pathogenic powdery mildew fungus. Cell Microbia18:1009- . Escola, J. M., MJ. Kleijmeer, W. Stoorvogel, J.M. Griffth, O. Yoshie, and H.J. geuze. (1998). Selective enrichment ot` tetraspan proteins on the internal vesicles of multivesicular endosomesand 011 exosomes secreted by human B- . Buschow, S.I., B.W. vanBalkom, M. Aalbcrts, A.J. Heck, M. Wauben, and W.

stoorvogel (2010). MHC class II-associatedproteins in B-cell exosomes and 25418.61 potential functional implications for exosome biogenesis. Immunol. Cell Biol. 88:851-856 Simpson RJ, Lim JW, Moritz RL, Mathivanan S. (2009) Exosomes: proteomic insights and diagnostic potential. Expert.Rev.Prote0mics.; 6:267- Gogolak P, Rethi B, Hajas G, Rajnavolgyi (2003) E. Targeting dendritic cells Vermeulen, P. B., Verh0even, D., Hubens, G., Van Marck, E., Goovaerts, G., Huyghe, M., De Bruijn, E.A., Van Oosterom, A.T. and Dirix, L.Y. (1995).

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Claims (1)

REQUESTS . It is a product that can be used in cancer treatment and its feature is; It contains plant exosomes. . It is a product mentioned in claim 1 and its feature is; The plant exosome source is wheatgrass. . It is a product mentioned in the claim and its feature is; The vegetable exosome source is garlic. . It is a product mentioned in claim 1 and its feature is; The source of herbal exosomes is ginger. . It is a product mentioned in Claim 1 and its feature is; The plant exosome source is composed of wheatgrass-ginger-garlic combination. It is a product as in any of the claims 1-5 and its feature is; it is wound healing. It is a product as in any of the claims 1-6 and its feature is; It is in the form of serum, syrup, tablet, medicine, gel, cream. It is a product as in any of the claims 1-7 and its feature is; It is effective in prostate cancer, breast cancer, kidney cancer, pancreas, liver, bone, skin, brain, lung, lung membrane, uterus, ovary, stomach, intestine, bladder, blood, lymph, thyroid cancer types.